It is estimated that more than 5 million Americans suffer from heart failure. The economic cost to US society for heart disease was almost $40 billion per in the year 2009. Currently, people with late-stage heart failure have only two treatment options, a heart transplant or implantation of a mechanically-assisted heart device but in the US, just over 2,000 hearts/year are available for transplantation. Heart diseases related to heart muscle failure or heart muscle weakening are treatable with drugs or devices such as defibrillators, pacemakers or implanted pumps. However, in heart attacks, when heart muscle cells die, transplantation becomes the only option because cardiomyocyte regeneration in the human heart is generally very limited. Current approaches used or in clinical trials are not designed to regenerate heart muscle but rely on improving remaining heart function. In the case of stem cell (SC) therapies in clinical trials, beneficial effects are due to paracrine signals from transplanted cells or persistence as vascular endothelial cells. Because of the large heart disease patient population, an intense effort to develop myocardial cell replacement therapies is underway. Production of cardiomyocytes in a biotech sense is a very important goal that would have considerable applications in both drug discovery and heart failure treatment. However, despite progress, increasing the efficiency of stem or progenitor cells to become human cardiomyocytes has been very challenging. The main problem with increasing the yield of cardiomyocytes is the lack of effective ways to induce ESCs to afford cardiomyocytes involved in cardiogenesis. A critical issue is the low yields of cardiomyocytes from in vitro differentiation processes. The need is to produce human cells that mimic the cardiac cell's response and physiological behavior in an efficient and cost-effective manner is paramount. The ability to differentiate SCs into cardiac cells on a large biotech scale will lead to several advances. First, technology for large quantities of cells will be available for transplantation purposes. Second, CROs and Big Pharma will have large numbers of cells available for drug safety evaluation. An economically viable biotechnological process using readily available and inexpensive differentiation agents is needed. Herein, we propose to use a powerful combination of high content and high throughput cellular assays and dynamic medicinal chemistry to develop pure, easy to make, small molecule "toolbox" compounds to promote the induction of hESCs that will differentiate into cardiomyocytes. Promising cardiomyocyte differentiation agents (i.e., compounds 1-3) have been identified and refinement and development of these agents is the focus of this proposal. The Specific Aims include: 1) Test 740 structurally related compounds to 1-3 as inducers of cardiomyocytes in a validated human ESC assay and 2) Test compounds of Aim 1 in validated counterscreens to test for selectivity and mode of action of cardiomyocyte differentiation. Successful completion of the proposed work will provide an inexpensive toolbox of reagents useful for the induction of cardiomyocytes from human ESCs of widespread utility. PUBLIC HEALTH RELEVANCE: In the future, human stem cell therapy will provide a way to regenerate damaged heart muscle cells for heart attack victims. Current therapies only improve heart function and what is needed is the generation of new heart muscle cells. The goal of our work is to use chemical biology to develop small molecule "toolbox" compounds that will stimulate stem cell differentiation and produce human cardiomyocytes. Ultimately, the results from this work will provide toolbox reagents for use to grow cardiomyocytes for use in a biotechnology process to treat heart disease and to improve the safety of human drugs in development.